A sole coupling of beta-adrenergic receptor (betaAR) to stimulatory G protein (Gs) to activate adenylyl cyclase has been considered as a "dogma" of betaAR signaling. However, our recent studies have demonstrated that pertussis toxin (PTX) treatment specifically potentiates the positive inotropic effect of beta2AR but not of beta1AR stimulation in rat ventricular myocytes, suggesting that beta2AR is coupled to a PTX-sensitive G protein(s) in addition to the established Gs (Xiao, et al Mol. Pharmacl. 1995; 45:322-9). A recently generated a2AR overexpression transgenic mouse (TG4) provides a unique opportunity to further explore the role of PTX-sensitive G proteins in a2AR stimulation. Previous studies in TG4 mice in vivo indicate that the baseline myocardial contractility is markedly increased over that in the wild type (WT) mice, and that a mixed (beta1&beta2) aAR agonist, isoproterenol (ISO) failed to further increase cardiac contractility (Milano. et al. Science 1994;264: 582-6). These resul have been interpreted to indicate that the baseline myocardial contractility in the TG4 mouse is at the maximal level, and that this is du to a greater number of a2ARs in the active form in the absence of agonists (i.e. the R* state). In this study, we have examined effects of beta2AR stimulation on intracellular calcium and contractility in single heart cells isolated from both TG4 and wild type (WT) mouse hearts. Under control conditions, the baseline contraction is 3.2-fold greater in TG4 cells over that of WT cells. beta2AR stimulation by zinterol has no effect on contraction amplitude in both WT and TG4 ventricular myocytes. However, PTX unmasks a potent positive inotropic effect and an increase in intracellular calcium after beta2AR stimulation by zinterol in both WT and TG4 heart cells and further increases the TG4 cell baseline contractility. Additionally, we provide evidence that beta2AR but not beta1AR agonists stimulate incorporation of the photoreactive GTP analog [alpha-32P]GTP azidoanilide into the alpha subunits of Gi (specifically Gialpha3) proteins; this is abolished by PTX pretreatment. These results demonstrate that both the spontaneously activated (R*) and agonist-activated beta2ARs are functionally coupled to Gialpha3, which, in turn, inhibit the cardiac inotropic response to beta2AR stimulation.